Identification of Three New Rugose Small Colony Variants from a Pseudomonas aeruginosa Biofilm

<i>Pseudomonas aeruginosa</i> is a Gram-negative, pathogenic, bacterium that produces biofilms comprising phenotypically distinct cell subpopulations. When separating and characterizing a single <i>P. aeruginosa</i> PA14 biofilm, three novel rugose small colony variants (RSCVs) (denoted RSCV_1, RSCV_2, and RSCV_3) were discovered. Characteristics of these stationary phase RSCVs differed between stationary phase wild-type (WT) PA14, between the PA14 biofilm subpopulations, and between the RSCVs themselves. The observed phenotypic changes in the RSCVs included differences in cellular morphology, exopolysaccharide production, biosynthesis of virulence factors, biofilm formation, and antibiotic tolerance. Stationary phase cell surface-associated molecules on the RSCVs were differently ionized as compared to WT PA14 using matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Many RNA transcripts were differentially expressed between the RSCVs and WT PA14 as well as between RSCV_1 and RSCV_3. DNA sequencing revealed single-nucleotide deletions and single-nucleotide polymorphisms (SNPs) among the RSCVs and between the RSCVs and WT PA14. The levels of the intracellular signaling molecule bis-(3',5')-cyclic-dimeric-guanosine monophosphate (cyclic-di-GMP) were higher in the RSCVs compared to WT PA14 and significantly lower in RSCV_3 as compared to both RSCV_1 and RSCV_2. The detected differences in the RSCVs have significant implications for biofilm production, antibiotic tolerance, and virulence.